PROJECT SUMMARY Paneth cells (PC) are specialized small intestinal epithelial cells that control microbial populations in the gut through the secretion of antimicrobial peptides (AMPs). Importantly, PC dysfunction is emerging as a driver of disease development in several gastrointestinal (GI) disorders, including Crohn?s disease, necrotizing enterocol- itis, and graft-versus-host disease. These disorders have limited therapeutic options, which often carry significant immunosuppressive and/or surgical risks. Ultimately, developing therapies to enhance PC function may provide a novel treatment strategy for these diseases. To accomplish this, further understanding of the regulatory mech- anisms responsible for normal PC biology is required. To this end, the present study seeks to build upon previ- ously published work from this applicant?s lab, which demonstrates that the enteric microbiota significantly influ- ence PC biology. Specifically, the objective of this application is to elucidate the precise mechanisms by which the resident gut microbiota exert their regulatory effects on PCs. The clinical rationale for this work is that the knowledge obtained will support the design of targeted strategies that augment PC function to putatively treat a spectrum of GI disease. The central hypothesis of this study is that the enteric microbiota increase PC census and promote AMP production via signaling through the subepithelial tissue (i.e. tissue underlying the intestinal epithelium). This hypothesis will be tested via two independent aims that respectively focus on host and microbial elements of PC regulation. In Aim 1, we seek to determine how specific subepithelial tissue factors are able to regulate PC function. Directed by our preliminary data, we will combine (A) targeted studies focused on IL22, with (B) a broader screen of rationally-selected additional molecules (Il17a, Retnlb, Jchain, S100a9, Cxcl5) to identify novel mechanisms by which the subepithelial tissue regulates PC biology. In Aim 2, the microbial side of these interactions will be explored. This will be facilitated by a novel PC-reporter mouse (Defa6-cre;td-To- mato) we have developed, which will be studied in both conventional and germ-free housing conditions. Specif- ically, these mice will be used to: (A) determine if bacteria implicated in the pathogenesis of Crohn?s disease (which has been linked to PC dysfunction) differentially regulate PC biology; and (B) ascertain if PC function can be enhanced using the established clinical probiotic, VSL#3 ? both at homeostasis and during inflammation. In combination, these aims are expected to identify host (Aim 1) and microbial (Aim 2) elements that can regulate PC function. The research proposed in this application is innovative because it explores the novel hypothesis that the subepithelial tissue is required to mediate microbial influences on PC biology. This substantially expands the conceptual framework of previous studies, which suggest that luminal bacteria directly interact with PCs to simulate antimicrobial function. The expected findings are significant because they will provide scientific justifi- cation for the development and future testing of novel agents that can clinically augment PC function. This has the potential to offer patients with limited therapeutic options new approaches for the treatment of their disease.